Introduction

Summary Introduction Procedures Results Discussion References

Despite the fact that small-molecular-weight GTPases regulate a diverse set of biological functions, the proteins share highly conserved guanine nucleotide binding domains. X-ray crystallographic studies of the elongation factor EF-Tu showed that Asp138 interacts with the guanine base of GDP by accepting hydrogen bonds from N1---H and the 2-NH₂ group (1, 2). In support of this model, point mutation of Asp138 to Asn138 greatly reduced the affinity of the protein for GDP but dramatically increased affinity for XDP (3). It is likely that Asn138 in mutant EF-Tu interacts with the base by donating a hydrogen bond to the 2-oxo group and accepting a hydrogen bond from the N1---H of xanthine (Fig. 1). Because Asp138 of EF-Tu is highly conserved, the aspartate-to-asparagine mutation in other GTPases is predicted to alter the nucleotide binding specificities of such mutants as well. Indeed, several GTPases with the aspartate-to-asparagine mutation have been reported to have high affinity and selectivity for binding xanthosine nucleotides (4-10).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Hydrogen-bonding schemes for purine ring binding of wild-type Rab5 and Rab5D136N. Left, hydrogen bonding between guanine and wild-type Rab5. Right, hydrogen bonding between xanthine and Rab5D136N.

Rab proteins are a family of Ras-like small-molecular-weight G proteins that are localized to distinct subcellular compartments and are implicated in intracellular membrane trafficking. One member of this family, Rab5, is localized on the plasma membrane, clathrin-coated vesicles, and early endosomes (11). Overexpression of Rab5 stimulates the rate of endocytosis and early endosome fusion (12-14). Rab5 is post-translationally modified with two geranylgeranyl (prenyl) groups on the two cysteines closest to its carboxyl terminus (15). This modification by prenylation is required for the membrane attachment and function of the protein (12, 16, 17).

Previous studies have shown that in vitro Rab5 prenylation in reticulocyte lysate was inhibited by nonhydrolyzable GTP analogs and that two point mutants of Rab5, which either preferentially bound GTP or had reduced GTPase activity, exhibited reduced rates of prenylation (18). These results indicate that only the GDP-bound form of Rab5 is the substrate for geranylgeranylation. Furthermore, when a cognate mutant of EF-Tu^D138N, Rab5^D136N, was constructed, its prenylation was dependent on the concentration of XDP instead of GDP, indicating that the nucleotide binding specificity was switched from guanosine to xanthosine nucleotides (9).

Given the possibility that nonhydrolyzable XTP analogs might inhibit prenylation of Rab5^D136N and allow studies of Rab5-dependent processes independently of other GTPases, we undertook the synthesis of XTP derivatives that would be expected to be insensitive to the XTPase activities of the mutant Rab5. We report a simple and superior method to prepare nucleoside triphosphate derivatives and its application to the synthesis of the xanthosine derivatives XMPPCH₂P and XMPPNHP. As anticipated, these compounds are potent inhibitors of XDP-dependent Rab5^D136N prenylation in reticulocyte lysate.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials. Xanthosine phosphates (XMP, XDP, and XTP) were purchased from Sigma Chemical (St. Louis, MO), and geranylgeranyl pyrophosphate was purchased from American Radiolabeled Chemicals (St. Louis, MO). All other chemicals were from Aldrich Chemical (Milwaukee, WI); DMF and N-methylimidazole were Sure-seal. TEAB was prepared by bubbling CO₂ gas through a mixture of water and freshly distilled triethylamine to pH 7.7. Glass double- distilled water was used throughout.

XMPPCH₂P. A solution of XMP sodium salt trihydrate (50 mg, 0.1 mmol) in water (1 ml) was passed through a 20- × 1-cm column of Dowex 50WX8 in the pyridinium form, and eluted with 2 column volumes of water. Tri(n-butyl)amine (48 µl, 0.2 mmol) was added to the eluate, followed by isopropyl alcohol until the mixture became homogeneous. The solution was concentrated under vacuum and lyophilized. The residue was dissolved in dry DMF (0.5 ml) in a 15-ml screw-capped centrifuge tube under N₂. A solution of triphenylphosphine (79 mg, 0.3 mmol), 2,2-dipyridyl disulfide (69 mg, 0.3 mmol), and N-methylimidazole [41 mg (40 µl), 0.5 mmol] in dry DMF (0.3 ml) was added. After 6 min, the N-methylimidazolide of XMP was precipitated by addition of cold dry diethyl ether (10 ml), and the mixture was centrifuged. The ether was decanted, and the solid was resuspended in 10 ml of ether, centrifuged and decanted again, and the residue was dried briefly in a gentle stream of N₂.

12

12

23

23

34

45

55"

5P1

45"

55"

XMPPNHP. A solution of tetrasodium imidodiphosphate (9.5 hydrate; 218 mg, 0.5 mmol) in ice cold water (2 ml) was passed through a column of Dowex 50WX8 in the H⁺ form and eluted with ice cold water until the eluate was neutral. Tri(n-butyl)amine (0.476 ml, 2.0 mmol) was added immediately to the ice cold eluate, and i-PrOH was added until the mixture became homogeneous. The solution was concentrated under high vacuum at room temperature and lyophilized. This tri(n-butyl)ammonium salt was dissolved in dry DMF (1 ml), and the solution was added to the N-methylimidazolide of XMP, prepared as described above (0.1 mmol). The mixture was vortexed until it became homogeneous (about 3 min). After 30 min, the reaction mixture was diluted with 20 ml of ice cold 0.1 M TEAB, loaded on a DEAE-Sephadex column (2 × 20 cm), and eluted at 4° with a linear gradient of 0.1-1.0 M TEAB during 16 hr at a flow rate of 2.67 ml/min. Fractions 74-86 (16 ml each) were combined and evaporated under high vacuum at room temperature. The residue was dissolved in a small amount of water and lyophilized to give XMPPNHP as the triethylammonium salt (21.6 mg, 35% yield). This product was converted to the sodium salt by passing an aqueous solution through a column of Dowex 50WX8 in the Na⁺ form. [¹H NMR (D₂O)  7.92 (s, 1 H, 8-H), 5.75 (d, 1 H, H-1; J₁₂ = 5.9 Hz), 4.49 (t, 1 H, H-2, J₁₂  J₂₃  5.6 Hz), 4.41 (dd, 1 H, H-3, J₂₃ = 5.3 Hz, J₃₄ = 3.6 Hz), 4.16 (m, 1 H, H-4), 4.09 (ddd, 1 H, H-5, J₄₅ = 3.1 Hz, J_55" = 11.7 Hz, J_5P1 = 6.7 Hz), 4.03 (ddd, 1 H, H-5", J_45" = 3.3 Hz, J_55" = 11.7 Hz, J_5"P1 = 4.7 Hz); ³¹P NMR (D₂O)  0.08 (d, P³, J_P2P3 = 4.4 Hz), 6.95 (dd, P², J_P1P2 = 20.8 Hz, J_P2P3 = 4.6 Hz), 9.94 (m, ¹H dec., d, P¹, J_P1P2 = 20.9 Hz). HR FAB mass spectra calculated for [C₁₀H₁₂N₅O₁₄P₃Na₄+H-2Na], 565.9455; found, 565.9467.

In vitro prenylation of Rab5^D136N. A point mutant of Rab5, Rab5^D136N, was constructed in the transcription competent vector, pAGA, as described previously (9). Rab5^D136N was synthesized in vitro using rabbit reticulocyte lysate programmed with the Rab5^D136N transcripts in the presence of [³⁵S]methionine (9.7 × 10¹⁷ cpm/pmol) according to the method described in Ref. 19. [³⁵S]-labeled Rab5^D136N (1 nM) was prenylated in 10 µl of geranylgeranylation mixture (12 mM Tris, pH 7.5, 0.6 mM dithiothreitol, 3 mM MgCl₂, 45% rabbit reticulocyte lysate, 20 µM geranylgeranyl pyrophosphate) containing various amounts of xanthine nucleotides (XDP, XTP, XMPPCH₂P, or XMPPNHP). Reactions were conducted at 37° for 1 hr; parallel reactions on ice were used as controls for nonmodified protein. The reactions were stopped by addition of Laemmli sample buffer, and the entire reaction mixtures were electrophoresed through a urea/acrylamide gradient sodium dodecyl sulfate gel (19). The gel was processed for fluorography, dried, and exposed to film. For quantitative analysis of the extent of protein prenylation, gel slabs corresponding to each of the ³⁵S-labeled protein bands were sliced off using the fluorograph as a guide. The gel slices were solubilized in 30% H₂O₂ by incubation at 65° overnight, and 20 ml of Scintiverse II (Fisher Scientific, Fair Lawn, NJ) was added for counting. The percentage of prenylated Rab5^D136N in each reaction was calculated as the ratio of radioactivity in the prenylated (lower) band to the sum of radioactivity in the prenylated and unprenylated (upper) bands after background was subtracted from each band. Apparent K_D values were calculated from IC₅₀ values as described (20).

	Results

Summary Introduction Procedures Results Discussion References

N-Methylimidazolides in nucleoside triphosphate synthesis. The most widely cited procedure for synthesis of nucleoside triphosphates and their modified derivatives involves activation of the monophosphates to the corresponding imidazolides with carbonyldiimidazole, followed by condensation of the activated intermediate with pyrophosphate or modified pyrophosphates (21). This procedure is lengthy (2-4 hr for activation and 12-24 hr for the condensation step) and results in partial modification of the 2- and 3-hydroxyl groups of ribonucleotides to 2,3-cyclic carbonates (22). These disadvantages prompted a search for a new method applicable to nucleoside triphosphate synthesis. It is known that N-methylimidazolides of carboxylic acids are more active acylating agents than the imidazolides (23), and it was reported recently that N-methylimidazolides of nucleoside monophosphates and oligonucleotides reacted readily with amines to give phosphoroamidates in high yields (24). A test reaction to apply this activated intermediate to nucleoside triphosphate synthesis was performed with N²(p-butylphenyl)-deoxyGMP (25). Reaction of the monophosphate with triphenylphosphine, 2,2-dipyridyl disulfide, and N-methylimidazole in DMF gave, after precipitation with diethyl ether, an product identified by ¹H and ³¹P NMR as the N-methylimidazolide. Stirring of the solid with a solution of tetrabutylammonium pyrophosphate in DMF for 5 min gave, after ion exchange chromatography, a 65% yield of N²(p-butylphenyl)-deoxyGTP, identical with a sample prepared by the classical imidazolide method (25) (results not shown).

Effect of XTP Derivatives on Prenylation of Rab5^D136N. To examine the properties of the XTP derivatives, their ability to inhibit prenylation of Rab^D136N was tested. Previous studies (18) have demonstrated that Rab geranylgeranyl transferase preferentially recognizes Rab proteins in the GDP-bound state and that nonhydrolyzable GTP derivatives block prenylation of Rab5 in vitro. Using an in vitro prenylation assay, we have also shown that modification of Rab5^D136N is dependent on XDP rather than GDP, demonstrating the conversion of nucleotide-binding specificity in this mutant (9). This XDP-dependent modification of Rab5^D136N is confirmed as illustrated in Fig. 2A. The post-translational modification of Rab5 is demonstrated by the shift to a higher mobility isoform on urea-gradient sodium dodecyl sulfate/polyacrylamide gels (19). As shown in Fig. 2A, Rab5^D136N is not prenylated in the absence of XDP, but it becomes completely modified when incubated at 37° for 1 hr in the presence of 100 µM XDP.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of xanthine nucleotides on prenylation of Rab5D136N. 35S-Labeled Rab5D136N was synthesized as described in Experimental Procedures. The protein (1 nM final concentration) was incubated in reticulocyte lysate for 1 hr on ice or at 37° in the absence or presence of 100 µM XDP (A) or in the presence of 100 µM XDP and increasing concentrations of XTP, XMPPCH2P, or XMPPNHP (B). Reactions were stopped and electrophoresed as described (19).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Inhibition of Rab5D136N prenylation as a function of xanthine nucleotide concentration. , XTP; , XMPPCH2P; , XMPPNHP. Percentage of Rab5D136N prenylation, determined as described in Materials and Methods, is plotted against xanthine nucleotide concentration. A representative experiment from four repeats is shown.

	Discussion

Summary Introduction Procedures Results Discussion References

A rapid and convenient method to prepare nucleoside triphosphates and ,-modified derivatives has been described. Preparation by a reported method (24) of N-methylimidazolides of nucleoside monophosphates and their isolation by diethyl ether precipitation avoids both side reactions and imprecise control of reaction conditions. Coupling of the intermediate with pyrophosphate or pyrophosphate analogs gives the triphosphate derivatives rapidly and in good yields.

The xanthosine 5-triphosphate derivatives XMPPCH₂P and XMPPNHP were synthesized by the new method and characterized with respect to standard spectroscopic properties (see Experimental Procedures). Both compounds blocked the XDP-dependent prenylation of Rab5^D136N in a concentration-dependent manner. The apparent K_D values were surprisingly similar, considering that analogous GTP derivatives (guanosine 5-(,-methylene)triphosphate and guanosine 5-(,-imido)triphosphate) typically differ significantly in their affinity to GTP-binding proteins; the imido compound has higher affinity than the methylene compound (26). Our observation that both modified triphosphates displayed persistent inhibition of Rab5^D136N prenylation but that XTP, which is hydrolyzed by Rab5^D136N, did not (Fig. 2), strongly suggests that XMPPCH₂P and XMPPNHP are not subject to hydrolysis by Rab5^D136N.

GTPases play pivotal roles in a diverse set of important cellular functions. For many of these biological pathways, such as signal transduction, intracellular membrane trafficking, and protein synthesis, multiple GTPases are involved. Thus, it is difficult to dissect the role of GTP hydrolysis by an individual GTPase in the background of many GTP-binding activities. One approach has been to selectively mutate one GTP-binding protein to XTP-binding specificity by conversion of aspartate to asparagine in the conserved NKXD guanine nucleotide-binding motif. This point mutation has been shown to alter nucleotide-binding specificity of several GTPases, including EF-Tu (3), Ras (10), Rab5 (9), Ypt1 (8), HypB (6), FstY (7), and adenylosuccinate synthetase (5). All of these mutants display altered nucleotide-binding specificity and possess XTPase activity. Furthermore, as long as xanthine nucleotide is present, the interaction with regulatory factors and the biological function of the aspartate-to-asparagine mutants remain unaffected (3, 6-8, 10). Such mutants have proven to be powerful tools for studying the mechanism of wild-type protein function (4, 6-8, 27). Such nonhydrolyzable derivatives as XMPPCH₂P and XMPPNHP should prove to be valuable reagents for future studies of other XTP-binding mutants, at least in in vitro assay systems, with particular importance in discriminating the function of nucleotide hydrolysis in various biological pathways.